The nuclear pore complex: A protein machine bridging the nucleus and cytoplasm

Current Opinion in Cell Biology

Volumn 12, Issue 3, 2000, Pages 361-371

  Ryan, Kathryn J ^a   Wente, Susan R ^a  

^a WASHINGTON UNIVERSITY SCHOOL OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

NUCLEAR PROTEIN; NUCLEOPORIN;

CELL NUCLEUS MEMBRANE; CYTOPLASM; INTRACELLULAR TRANSPORT; MITOSIS; NONHUMAN; PRIORITY JOURNAL; REVIEW; STRUCTURE ACTIVITY RELATION; YEAST;

EID: 0034022647     PISSN: 09550674     EISSN: None     Source Type: Journal    
DOI: 10.1016/S0955-0674(00)00101-0     Document Type: Review

References (93)

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5. Nakielny S., Shaikh S., Burke B., Dreyfuss G. Nup153 is an M9 containing mobile nucleoporin with a novel Ran-binding domain. EMBO J. 18:1999;1982-1995. A short amino-terminal portion (amino acids 247-290) of Nup153 was isolated as a potential Kapβ2/transportin substrate. This peptide was able to function as a NLS when fused to a larger protein. Further characterization of Nup153-Kap interactions revealed that multiple Kaps could interact directly with different regions of Nup153. Although Nup153 is localized to the nuclear basket under steady state conditions, anti-Nup153 antibodies can be transported from the cytoplasm into the nucleus in the absence of new protein synthesis. This result implies that, like other Kapβ2 substrates, Nup153 shuttles between the nucleus and cytoplasm.
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9. Katahira J., Straber K., Podtelejnikov A., Mann M., Jung J.U., Hurt E. The Mex67p-mediated nuclear mRNA export pathway is conserved from yeast to human. EMBO J. 18:1999;2593-2609. Human TAP is the apparent orthologue of scMex67. Steady-state localization of TAP was primarily nuclear, with a fraction associating with the NPCs. In heterokaryon assays, TAP shuttled between the nucleus and cytoplasm. A two-hybrid screen identified Nup214 and a novel protein, termed hCG1, as potentially interacting with TAP. The interaction between Nup214 and TAP was confirmed with in vitro binding assays. Co-immunoprecipitation identified a second novel protein, p15, as interacting with TAP. Mex67 interacts physically with Mtr2, and together they are required for mRNA export [78]. Although neither TAP or p15 could rescue a mex67 or a mtr2 null strain, co-expressing TAP and p15 rescued both null cells. These results imply that the individual proteins in this mRNA transport complex have diverged in parallel so that the overall function of the complex is unchanged.
10. Strahm Y., Fahrenkrog B., Zenklusen D., Rychner E., Kantor J., Rosbash M., Stutz F. The RNA export factor Gle1p is located on the cytoplasmic fibrils of the NPC and physically interacts with the FG-nucleoporin Rip1p, the DEAD-box protein Rat8p/Dbp5p and a new protein Ymr255p. EMBO J. 18:1999;5761-5777. A high copy suppressor screen of gle1 identified three genes, NUP42/RIP1, DBP5, and GFD1, each capable of rescuing the temperature sensitivity in S. cerevisiae. In two-hybrid and in vitro binding assays, each of the suppressors demonstrated an interaction with the carboxy-terminal domain of scGle1. Localization of scGle1-GFP to the nuclear rim is reduced, but not eliminated, in a nup42 null strain, suggesting that Nup42 helps anchor scGle1 at the NPC. IEM of Protein A-tagged scGle1 and scDbp5 showed localization to the cytoplasmic fibrils and cytoplasm (Dbp5 only). Other groups localized endogenous scGle1 also to the nuclear face (DM Barry, SR Wente, unpublished data; MP Rout, personal communication). Nup42 is also found associated with the fibrils, as well as with the nuclear basket. Based on this localization data, Strahm et al. propose that these factors act in the terminal steps of mRNA transport. The authors also isolated a human hCG1 clone and showed it could functionally replace Nup42.
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14. Belgareh N., Snay-Hodge C., Pasteau F., Dagher S., Cole C.N., Doye V. Functional characterization of a Nup159p-containing nuclear pore subcomplex. Mol Biol Cell. 9:1998;3475-3492. The interactions of scNup159 with other Nups were investigated in a series of co-immunoprecipitation studies with Protein-A-tagged Nups from various yeast strains. Nup159 was isolated with the Nsp1/Nup82 complex [12]. The carboxy-terminal portion of Nup159 was required for interactions with both Nsp1 and Nup82. Although Nup159 and Nsp1 interacted independently of Nup82, the stable association of Nsp1 and Nup82 required Nup159. Localization of Nup159 to the NPC was lost upon temperature shift in a nup82 mutant in which Nup82 protein is degraded at the non-permissive temperature. These results suggest a hierarchy of assembly in the NPC where Nup82 anchors Nup159, which in turn provides a binding site for Nsp1.
15. Bucci M., Wente S.R. A novel fluorescence-based genetic strategy identifies mutants of Saccharomyces cerevisiae defective for nuclear pore complex assembly. Mol Biol Cell. 9:1998;2439-2461. A fluorescence-based screen for mutants defective in scNPC assembly was performed in a GFP-tagged nup49 strain. It was predicted that failure to assemble complete NPCs would result in the loss of GFP-Nup49 at the NE and diminished fluorescence intensity. The screen for such 'dim' mutants identified a temperature sensitive allele of nup57. In addition to Nup49, Nsp1 and Nup116 were also mislocalized in the nup57 mutant, whereas the localization of Pom152, Nic96, Nup82, Nup159 and Nup145 was not remarkably altered. This type of strategy may be a powerful tool for identifying how the NPC is assemble.
16. Marelli M., Aitchison J.D., Wozniak R.W. Specific binding of the karyopherin Kap121p to a subunit of the nuclear pore complex containing Nup53p, Nup59p, and Nup170p. J Cell Biol. 143:1998;1813-1830. NUP59 was identified in a synthetic lethal screen with pom152, and NUP53 was identified based on its sequence similarity to NUP59. Neither was essential for viability; however, both showed synthetic lethal interactions with the other Nups identified in the original pom152 screen. Isolation of a protein-A-tagged Nup53 from yeast extracts showed that Nup53 was in a complex with Nup59, Nup170 and Kap121. Using blot overlays, it was determined that Kap121 binds directly to Nup53. Weaker interactions between Nup59 and Kap95, Kap123 and Kap121 were also detected. This is one of the first examples of Kaps interacting with non-repeat containing Nups.
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22. Adam S.A. Transport pathways of macromolecules between the nucleus and the cytoplasm. Curr Opin Cell Biol. 11:1999;402-406.
23. Yaseen N.R., Blobel G. GTP hydrolysis links initiation and termination of nuclear import on the nucleoporin Nup358. J Biol Chem. 274:1999;26493-26502. Recycling of Kapβ1 from the nucleus back to the cytoplasm occurs in a complex containing RanGTP. Using in vitro assays, this Kapβ1/RanGTP complex interacted with RanBP1 homologous domains of Nup358/RanBP2, a component of the cytoplasmic fibrils. The interaction was sensitive to competition with excess RanBP1. EM localization of Kapβ1 to cytoplasmic fibrils in permeablilized cells also required Ran and was sensitive to RanBP1 competition. This strongly suggested the Kapβ1/RanGTP/Nup358 interactions occur in vivo.
24. Kehlenbach R.H., Dickmanns A., Kehlenbach A., Guan T., Gerace L. A role for RanBP1 in the release of CRM1 from the nuclear pore complex in a terminal step of nuclear export. J Cell Biol. 145:1999;645-657. An in vitro assay was used to identify cytosolic factors required for Crm1-mediated protein export. In permeabilized cells, preincubation with a Ran mutant unable to hydrolyze GTP blocked nuclear export and Crm1 accumulated at the cytoplasmic side of NPCs. Under these conditions, Crm1 associated with p62 and Nup214/CAN. Reconstitution experiments demonstrated that RanBP1 and the RanBP1 homologous domains of Nup358/RanBP2 were capable of dissociating Crm1 from the NPC even in the presence of mutant Ran. These results suggested that RanBP1 and Nup358 promote the release of Crm1 at the cyoplasmic fibrils in a terminal step of protein export.
25. Shah S., Forbes D.J. Separate nuclear import pathways converge on the nucleoporin Nup153 and can be dissected with dominant-negative inhibitors. Curr Biol. 8:1998;1376-1386. A short FG repeat region of Nup153 acts as a dominant negative inhibitor of Kapα/β1 (importinα/β) import; however, it has no effect on either Kapβ2 (transportin) or Crm1-mediated transport. A second fragment (amino acids 431-723) specifically blocked Kapβ2 transport, but had no effect on Kapα/β1 or Crm1 transport. This fragment assembled with Kapβ2 only in the absence of cargo, and association was sensitive to the nucleotide-bound state of Ran. These results indicate that although Nup153 is required for several different classes of transport, different receptors may use distinct, non-overlapping sites of interaction.
26. Shah S., Tugendreich S., Forbes D. Major binding sites for the nuclear import receptor are the internal nucleoporin Nup153 and the adjacent nuclear filament protein Tpr. J Cell Biol. 141:1998;31-49. Immunoprecipitation experiments from Xenopus egg extracts or extracts from assembled NPCs show that Kap/importin β co-isolates with Nup153 and Tpr, but not with p62, Nup93, Nup98 or Nup214. Using purified and recombinant proteins, direct binding of Kapβ and Nup153 was shown. No direct association of Kapβ and Tpr was observed. Kap/importin α was also present in the Kapβ-Nup153 or Kapβ-Tpr complexes; however, a NLS-cargo was only found in the Kapα/β/Nup153 complex. With Nup153 at the nuclear basket and Tpr at the intranuclear filaments, the authors propose a role for these interactions in the terminal steps of protein import.
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